Manipulating sorting signals to generate co-expression of somatostatin and eGFP in the regulated secretory pathway from a monocistronic construct.

Targeted overexpression of biologically active peptides represents a powerful approach to the functional dissection of neuroendocrine systems. However, the requirement to generate separate, biologically active and reporter molecules necessitates the use of internal ribosome entry site (IRES) technology, which often results in preferential translation of the second cistron. We report here a novel approach in which the proteolytic processing machinery of the regulated secretory pathway (RSP) has been exploited to generate multiple mature proteins from a monocistronic construct that encodes a single precursor. This was achieved by duplication of the pre-pro cleavage sites in pre-prosomatostatin cDNA. The duplicated site included 10 flanking amino acids on either side of the Gly-Ala cleavage position. This enabled the incorporation of a foreign protein-coding sequence (in this case, enhanced green fluorescent protein (eGFP)) between these sites. The pre-eGFP-prosomatostatin (PEPS) construct generated co-localized expression of fully processed eGFP and somatostatin to the RSP of transiently transfected AtT20 cells. This approach represents an advance upon bicistronic and other extant approaches to the targeting of multiple, biologically active proteins to neuroendocrine systems, and, importantly, permits the co-expression of fluorescent markers with biologically active neuropeptides. In this study, our demonstration of the fusion of the first 10 amino acids of the prosomatostatin sequence to the N-terminus of eGFP shows that this putative sorting sequence is sufficient to direct expression to the RSP.

Optimisation of methods for selecting candidate genes from cDNA array screens: application to rat brain punches and pineal.

DNA arrays are potentially powerful experimental tools within neuroscience but application of this technology to in vivo paradigms may, in practice, be limited by the sensitivity of transcript detection and inter-screen variation. Here we describe the use of brain punch micro-sampling, used in combination with commercially available cDNA arrays, for profiling brain gene expression in a mutant strain of rat (GAERS model of absence epilepsy). Furthermore, we describe a multi-step optimisation of analysis methods which provides for improved sensitivity and absence of bias in the selection of candidate genes which may be differentially expressed in the mutant. Our method has been validated through application to a second paradigm, rhythmic gene expression in the rat pineal gland. Our experimental design, and analysis method should therefore be generally applicable to subtle discriminations of transcript abundance within discrete brain areas.